Human growth hormone (hGH) is a key factor in the regulation of certain physiological processes, such as growth and differentiation of muscle and bone cells. The hGH signal is mediated by homodimerization of two identical human growth hormone receptors (hGHR), Wells, et al, Annu. Rev. Biophys. Biomol. Struct., 22, 329-51 (1993). To accomplish this task, hGH interacts with each receptor using two distinct binding epitopes (hGH site 1 and 2), that both bind at the domain interface of the extracellular part of the receptor, deVos et al, Sciences, 255, 306-12 (1992) (FIG. 1). However, an important and hitherto unresolved question is if the receptor undergoes conformational changes to facilitate hGH binding. hGH binds to the hGHR but also to its naturally occurring soluble binding protein (hGHbp), Bauman et al, J. Clin. Endocrinol. Metab., 78, 1113-8 (1994). Heterologously expressed variants of hGHbp are therefore commonly used to study the extracellular events in receptor dimerization, Fuh et al, J. Biol. Chem., 265, 3111-5 (1990). Moreover, in a zinc dependent interaction, hGH also can bind to the human prolactin receptor (hPRLR), Cunningham et al, Science, 250, 1709-12 (1990).
It would be highly desirable to obtain a model to study the structural requirements for the transition of the extracellular part of the human growth hormone receptor from its free, unliganded state to its homodimeric state complexed with a ligand. Such studies could potentially lead to a more detailed understanding of the binding conditions specific for hGHR and would constitute important means in obtaining new drug candidates with ligand activity. However, it has so far been impossible to perform binding studies of hGHR in crystalline form without having the receptor molecule bound to a natural ligand. The reason is that both hGHR and other cytokine receptors have been found difficult to crystallize in their unliganded form, since they most likely contain domains and/or loop regions that are flexibly connected which contributes to a disordered state which obstructs crystallization.
It is the object of the present invention to provide modified extracellular domains of cytokine receptors which solve the described problems and which are capable of being crystallized with conventional methods. Furthermore, it is an object of the present invention to provide crystals of modified extracellular receptor proteins which are exceptionally useful for binding studies with small molecules that, in the absence of the natural ligand, are free to interact with the receptor binding surfaces.